Glyphosate tolerant plants are known in the art and well studied in the past two decades. Glyphosate is an herbicide inhibiting EPSPS which is an enzyme whose activity is upstream of the aromatic amino acids pathway leading to the synthesis of the amino acids tyrosine, tryptophan and phenylalanine. Since glyphosate is a systemic total herbicide, tolerance in the plant when the herbicide is sprayed under usual agronomic conditions may only be achieved by genetic modification of all cells of the plants with an heterologous gene coding for a glyphosate insensitive EPSPS enzyme, either mutated or selected from microorganisms known to have evolved such insensitive EPSPS enzyme.
Glyphosate insensitive EPSPS, gene constructs and plants transformed with said gene constructs are disclosed inter alia in EP 507 698, EP 508 909, U.S. Pat. No. 4,535,060, U.S. Pat. No. 5,436,389, WO 92/04449, WO 92/06201, WO 95/06128, WO 97/04103, WO 2007/064828 and WO 2008/100353, and in references cited herein.
The biophysical characteristics of the EPSPS protein are essential to achieve a good level of tolerance to glyphosate. However, the choice of regulatory elements providing an adequate expression level of the insensitive protein in the plant is also important, as well as the selection of a transformation event, corresponding to a stable line with a stable and limited number of copies of the gene being inserted in the genome of the plant, as well as its stability in the locus where the gene has been inserted is also important to obtain glyphosate tolerance at a commercial level, sufficient for the plant to be used for the preparation of seeds to be planted in a field with a level of tolerance to glyphosate under agronomic conditions sufficient to allow use of the herbicide at effective concentrations to kill the weeds without affecting growing conditions and yields of the crop transformed with the gene encoding EPSPS protein.
Transformation events selected for the preparation of commercial varieties of glyphosate tolerant maize (corn) are known in the art, particularly disclosed in U.S. Pat. No. 6,040,497 and EP 1 167 531.
These varieties of the first generation used for the preparation of commercial plants currently used in the field have some drawbacks.
The event GA21 disclosed in U.S. Pat. No. 6,040,497 comprise multiple copies of a gene construct comprising a rice actin promoter and intron, a sequence coding for an optimized transit peptide, as disclosed in EP 505 909 and a sequence coding for a mutated plant EPSPS comprising two mutations as disclosed in WO 97/04103. The commercially required level of tolerance in the transformation event is obtained with a complex transit peptide and multiple copies of the chimeric gene construct.
The event NK603 disclosed in EP 1 167 531, is also a complex event with the combination of two gene constructs in one locus. The first gene construct comprises a rice actin promoter and intron, with a sequence coding for an Arabidopsis EPSPS transit peptide and a sequence coding for a type II EPSPS resistant to inhibition by glyphosate, isolated from Agrobacterium strain CP4. The second gene construct comprises the CaMV 35S promoter and the rice actin intron, with a sequence coding for an Arabidopsis EPSPS transit peptide and a sequence coding for a type II EPSPS resistant to inhibition by glyphosate, isolated from Agrobacterium strain CP4.
There is a need for a new generation of transformation events allowing a high glyphosate tolerance to maize (corn) plants grown under agronomic conditions with a single copy of the foreign gene construct in the plant genome.